Proteinase inhibitors are of potential therapeutic importance as regulators of tumor cell mediated enzymatic degradation of the extracellular matrix. The classes of proteinases most commonly associated with tumor invasion are: metalloproteinases, plasminogen activators and cathepsins. Tissue inhibitors of metalloproteinases (TIMP) and plasminogen activator inhibitors (PAI) have been shown to suppress tumor cell invasion in vitro and metastatic propensity in vivo. Recently a trypsin-like enzyme has been purified from ovarian cancer and shown to activate other proteinases associated with malignancy, such as collagenases and plasminogen activators. Endothelial cells are known to express high constitutive levels of TIMP and PAI in vitro. The present study is mainly focused on a search for new endothelial proteinase inhibitors and their possible role in blocking tumor dissemination. Conditioned medium from human umbilical vein endothelium was screened for trypsin inhibitors using reverse zymography technique. There is preliminary evidence for the expression of trypsin inhibitors by endothelial cells. Four trypsin resistant proteins of 15, 21, 31, and 40kDa were isolated from endothelial cell conditioned medium by trypsin affinity chromatography. Considering the possible role of a tumor derived trypsin-like enzyme(s) as activator(s) of the major matrix degrading proteinases, trypsin inhibitors expressed by endothelial cells may block their action and thus interfere with the proteolytic cascade involved in tumor cell invasion.